Many analytical methods are directed towards the determination of a family of closely related compounds. These families of compounds may arise in a test sample by a variety of mechanisms. For example, manufacturing operations often yield in addition to the desired product, a number of analogues, homologues or other derivatives. Further, families of compounds may be produced by the metabolic activity of living organisms on substances introduced into or produced within the organism.
It is frequently desired to determine all of the members of a family of compounds. For example, if the family members have similar biological activity it would be clinically useful to determine the concentration of the entire family. Similarly, a single compound may be clinically significant but available test samples may not contain a representative quantity of the compound because it has already been metabolically converted into other substances. Further, since precursors may be readily converted into the substance of interest in vivo it may be clinically useful to assay the precursors as well as the substance of interest. Thus from the standpoint of biological activity it may be most instructive to assay an entire family of metabolic relatives of the target compound. This is particularly the case with drugs and naturally occurring steroids which are commonly subject to hydrolysis, condensations and redox conversions as part of the normally occurring metabolism of the body. Estriol assays are an example.
Estriol, an estrogen produced by the placenta, is assayed to monitor fetal viability. As a practical matter the estriol must be measured in the mother's plasma or urine. However, the mother's liver anabolizes estriol to various sulfate and glucuronide conjugates; only a small proportion of estriol is found in the plasma or urine. Thus, an adequate measure of placental estriol elaboration must include a reconversion of the estriol anabolic conjugates to estriol. While it is possible to determine the amount of each conjugate in the estriol family, it is more convenient to simply convert the conjugates into estriol. Then a single assay will measure the total amount of estriol produced by the placenta.
Receptor binding assays are frequently used to determine families of compounds. Receptor binding assays all exploit the capacity of a receptor, usually a protein such as an antibody, to recognize and reversibly bind an analyte. For example, in the commonly encountered competitive immunoassay technique, a receptor antibody is equilibrated with the analyte and a labeled analogue of the analyte. The labeled analogue usually contains a covalently bound moiety, e.g., a radioisotope or enzyme, which is foreign to the other constituents of the assay. The labeled analogue, however, is labeled so that it is recognized and bound by the receptor with approximately the same affinity as the analyte. A deficiency of receptor binding sites compared to the total of labeled plus sample analyte binding sites is provided, thus forcing the labeled and sample analytes to compete for a limited number of receptor sites. Since the labeled and sample ligand have an approximately equivalent affinity for the receptor, the total amount of labeled analyte bound to the receptor will be inversely proportional to the amount of sample analyte. The receptor-bound analyte, both labeled and unlabeled, can then be separated from the remaining unbound analyte and the quantity of receptor-bound label compared with a standard curve to arrive at a quantitative measure of the sample analyte.
A principal difficulty in assaying a family of compounds by a receptor binding method is that it is not practical to prepare a receptor which indiscriminately binds all members of a family of compounds with the same affinity. While an antibody raised by known techniques against a first member of the family may indeed bind, i.e., cross-react with other members of the family, the antibody will rarely exhibit the same affinity for each member. The result is that some members will be preferentially bound at equilibrium over others. This will seriously distort the results of any assay using such a receptor. For example, it would be impossible to determine from a single receptor assay whether an elevated total for the family was a function of either a predominant proportion of high affinity analyte or higher concentration of low affinity analyte. Of course, a combined assay for a family of compounds is clearly impossible where the receptor fails to cross-react at all with some of the family members. One must then go to the expense and difficulty of conducting a plurality of determinations. Accordingly the nature of the receptors used in receptor binding assays makes it particularly desirable to convert all of the family members either to one of the family members or to a single compound not one of the family members.
Such a conversion has been incorporated by the prior art into a method for the determination of estriol. Free estriol and the estriol conjugates estriol-16-glucuronide, estriol-3-sulfate-16-glucuronide, estriol-3-sulfate and estriol-3-glucuronide are a family of estriol and its anabolites that are of clinical interest. The prior art method for the determination of this family comprises incubating the test sample with enzymes capable of hydrolyzing the glucuronide and sulfate conjugates to estriol. When the conversion to estriol is complete, the amount of estriol in the hydrolyzed sample is then determined by competitive radioimmunoassay. See, for example, Levitz et al and Horecht et al in "Steroids" at 27:287-294 (1976) and 25:587-590 (1975), respectively.
In all of the prior art methods for estriol and its conjugates of which applicants are aware the enzyme hydrolysis has been allowed to go to completion before the radioimmunoassay is conducted. The reasons for this are believed to be a concern that the enzyme incubation and the radioimmunoassay will mutually interfere.
First, the pH commonly employed for enzyme hydrolysis of estriol glucuronides and sulfates is about from 4 to 6, generally about 5. However, the pH at which antibody binding of haptens is ordinarily considered optimal is about from pH 7 to 8. Further, the enzyme preparations may contain proteases; these clearly could be deleterious to antibodies, particularly if the antibodies were exposed to the proteases throughout the enzyme incubation period.
Second, it is known that estriol antibodies cross-react with such estriol conjugates as estriol-3-sulfate and estriol-3-glucuronide. See Katagiri et al, "Steroids" 24:225-239 (1974). Yet these same conjugates must be enzyme hydrolyzed to estriol if the assay is to be accurate. Since the estriol antibody binds to these estriol conjugates it could be expected that the antibody would sterically hinder access of the enzyme to the conjugate. This could of course prevent the hydrolysis of the estriol conjugates entirely or, at the least, could excessively extend the period for their hydrolysis. In this connection, see U.S. Pat. Nos. 3,852,157 and 3,935,074, W. German Offenlegungsschrift 2,618,511 and French Patent Number 2,373,063.
This prior art procedure, however, is disadvantageous in several ways. First, the enzyme composition in commercial estriol assay kits must be separately packaged from the estriol antibody. This, of course, increases the cost of the kits. Second, the conduct of the prior art assay is complicated by the need to separately add the enzyme composition and to time the enzyme incubation period. This introduces additional sources of error into the assay. Third, the time for performing the assay is extended by the period of the enzyme incubation or the radioimmunoassay, whichever is shorter. This extended period of at least one hour is a serious impediment to the prompt conclusion of the determination.